Project Summary The overall goal of this renewal application is to develop modular microfluidic approaches to quantitatively study cell-to-cell interactions on 3D scaffolds. Most of the reported technology concerning microchip-based cell culture involves culturing of cells on a flat, 2D substrate. In many cases, these do not fully mimic the 3D cell growth that occurs in vivo. Additionally, most of the current technology lacks efficient online analytical schemes for real-time observation of important molecules released from the cells. To address these issues, in Aim #1, we propose to create a scalable and multiplexable microfluidic device for 3D cell culture. Polystyrene film coated with fibrous scaffolds (that act as 3D cell culture matrix) will be laser cut into inserts and integrated into a microfluidic device for flow-based cell studies. We also propose to develop a wall-tube electrochemical microfluidic device with pillar electrodes modified by Pt-black and Nafion for sensitive and selective NO detection, which can be coupled with the 3D cell culture device in a modular fashion for online NO quantitation. The effects of fibrous scaffolds on nitric oxide (NO) release from endothelial cells under shear stress will be studied. In Aim #2, the insert-based 3D cell culture device will be utilized for macrophage studies. The inserts are made by depositing a layer of fibrous scaffold on the polystyrene film. The fiber size, pore size and chemoattractant administration will be optimized to facilitate cells to infiltrate through the fibrous scaffold to form a true 3D tissue mimic. Multiple scaffolds can be inserted into the same device so that a macrophage laden insert can be removed after cell stimulation, with the various inserts being analyzed by techniques such as DNA quantitation (as a measure of cell amount), confocal microscope and cell membrane mapping. For example, the cells on an insert can be labeled by antibodies linked with fluorophores for quantitation of membrane markers specific to different macrophage phenotypes. We also propose to develop a PDMS device using 3D-printed sacrificing molds, which can be connected to the cell device for online quantitation of macrophage related nitrite (a biomarker of the pro-inflammatory M1 phenotype) in near real time. In Aim #3, the interaction between macrophages and endothelial cells in atherosclerosis will be mimicked by integrating the two cell types in one device coupled with downstream NO quantitation. The hypothesis that overproduction of NO by lesional endothelial cells may initiate inflammatory responses of macrophages, which in turn can cause further endothelium injury will be tested on the modular device. Overall, we will develop an innovative approach for 3D cell culture, which represents a new and versatile way for in vitro cell studies that better mimic in vivo systems. We will also create new quantitation modules for online quantitation of molecules of interest. This configuration may be adapted for any adherent cell type and easily adopted by other researchers. We will show two cell types integrated together; but more cell types can be included.